Glucose metabolism stimulates biphasic insulin secretion from pancreatic B-cells. It also alters islet electrical activity causing oscillations in membrane potential and elevation of cytosolic free Ca2+. The basis for the two phases of insulin secretion and the link between metabolic and electrical events are unknown. A model is presented in which intracellular mediation of first phase insulin secretion is a response to oscillations of the ATP/ADP ratio, initiating electrical/secretory events by transmitting metabolic changes to the plasma membrane. The model also suggests that second phase insulin release is mediated by a malonyl CoA-induced switch of energy production from fatty acids to glucose which leads to production of diacylgycerol and activation of protein kinase C. The goals of this proposal are to continue to develop and test this mechanistic model of fuel-induced insulin release in several ways. First, since the earliest electrical event in secretion is closure of ATP-sensitive K+- channels, we plan to determine the precise range of ATP/ADP ratios that affect this channel in order to find out if it is regulated at physiologically relevant ratios. Second, we plan to directly measure the ATP/ADP ratio and oxygen use in single stimulated islets to determine whether metabolic oscillations occur. Third, we plan to monitor cytosolic free Ca2+ in single cells to assess the effects of graded stimulation on this parameter which we expect will oscillate with increasing metabolic flux. We propose to evaluate the switch in islet metabolism from free fatty acids to glucose as its primary energy source. The results of this switch will be evaluated in two ways. First, we will measure the changes in the acyl CoA profile that accompany stimulation. Second, we will characterize the effect of acyl CoA compounds, particularly those that change during stimulation, on cellular Ca2+ handling in permeabilized insulinoma cells. Finally, we will test predictions made by the composite model on insulin secretion in single isolated pancreatic islets. This will be done by imposing variations in the intracellular metabolic couples including the ATP/ADP, NAD/NADH and acyl CoA/CoASH ratios. The effect of these perturbations on insulin secretion will be compared with predictions from the model.